The general view on Rhizobium chitolipooligo-saccharides (CLOS) is that they are made in very low levels as diffusible molecules and are primarily secreted by the bacteria where they interact with the host. However, the structural and predicted physicochemical properties of these amphiphilic molecules led us to postulate that they should normally be targeted to bacterial membranes after synthesis. Thus, we analyzed membrane lipid extracts of R. leguminosarum bv. trifolii, wild-type strain ANU843 cells and the corresponding culture supernatants for CLOS-type glycolipids. Fractionation of the membrane extracts from pelleted cells led to the isolation of a diverse family of CLOS in high yield, whereas all attempts to isolate CLOS from the corresponding culture supernatant failed. Structural analyses reveal that the membrane CLOS of ANU843 consist of a complex mixture of O-acetylated or non-O-acetylated N-acylated chito-tri-, -tetra-, and pentasaccharides. cis-Vaccenic acid was the predominant acyl substituent (>70%), but several other saturated, unsaturated, and 3-hydroxy fatty acids were found in the CLOS glycolipids. Membrane accumulation of CLOS in ANU843 is promoted by the presence of 4 ,7-dihydroxyflavone and pSym nod genes. These results indicate that, a very diverse family of CLOS Nod factors accumulate primarily in bacterial membranes of wild-type Rhizobium.